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Team:TU Delft/Project/rbs-characterization/parts
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<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html> | <html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html> | ||
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BBa_I13401 was PCR amplified using the universal primers G00100 and G00101. The purified PCR product was assembled into one of the Anderson RBS plasmids provided in the distribution plates by means of 2-way ligations. | BBa_I13401 was PCR amplified using the universal primers G00100 and G00101. The purified PCR product was assembled into one of the Anderson RBS plasmids provided in the distribution plates by means of 2-way ligations. | ||
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The cells were cultured over 18 hours in 96-well plates using a Gen5 fluorescence and absorbance plate reader, LB with ampicillin was the culture medium used in these experiments. Only the results obtained during exponential growth phase were taken into account. | The cells were cultured over 18 hours in 96-well plates using a Gen5 fluorescence and absorbance plate reader, LB with ampicillin was the culture medium used in these experiments. Only the results obtained during exponential growth phase were taken into account. | ||
| - | + | <html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html> | |
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== Parts == | == Parts == | ||
Our constructs used for measurements: | Our constructs used for measurements: | ||
Revision as of 14:58, 22 October 2010
BioBricks, the making of the RBS Characterization
BBa_I13401 was PCR amplified using the universal primers G00100 and G00101. The purified PCR product was assembled into one of the Anderson RBS plasmids provided in the distribution plates by means of 2-way ligations.
Transformation into competent Top10 E.coli cells yielded positives, as determined by fluorescence analysis.
The cells were cultured over 18 hours in 96-well plates using a Gen5 fluorescence and absorbance plate reader, LB with ampicillin was the culture medium used in these experiments. Only the results obtained during exponential growth phase were taken into account.
Parts
Our constructs used for measurements: <partinfo>K398500 SpecifiedComponents</partinfo>
<partinfo>K398501 SpecifiedComponents</partinfo>
<partinfo>K398502 SpecifiedComponents</partinfo>
<partinfo>K398503 SpecifiedComponents</partinfo>
<partinfo>K398504 SpecifiedComponents</partinfo>
Experimental Setup
We took five different RBS sequences from the Anderson RBS family (J61100, J61101, J61107, J61117, J61127). The standard RBS B0032 was used as a reference for our characterization.
All these RBS sequences were placed in front of the standard GFP coding sequence, so expression could be measured. The Biobricks generated in order to perform the experiments were: K398500, K398501, K398502, K398503, K398504. The general map of the construction is shown below, where the RBS is displayed in fucsia.
| Feature | Function |
| AmpR | Ampicillin resistance |
| B0015 | Transcriptional (double) terminator |
| B0062 | Transcriptional terminator |
| E0040 | GFP |
| G00000 | Standard prefix |
| G00001 | Standard suffix |
| G00100 | VF2 primer binding site |
| G00101 | VR primer binding site |
| J61100 | RBS Anderson family |
| J23100 | Promoter |
